PROJECT SUMMARY This new R01 application seeks to define mechanisms of intestinal mucosal repair after stem cell injury, investigating the role of two key signaling pathways Dll1/4-Notch signaling and IGF1-mTORC1 signaling for promoting crypt repair. Adult stem cells fuel the continuous renewal of the intestinal epithelium. Prevailing theory suggests that there are two stem cell populations: active stem cells (also termed crypt base columnar (CBC) cells) important for epithelial cell maintenance during homeostasis and facultative stem cells (also termed quiescent or reserve stem cells) important for replenishing CBCs during crypt recovery after injury- induced stem cell loss. Intestinal crypts exhibit remarkable plasticity, with various epithelial cells in the crypt capable of reprogramming to fill unoccupied niche spots to replenish the CBC stem cell pool. Mechanisms of crypt repair and facultative stem cell mobilization are currently poorly understood. This project aims to define key niche signals that drive the regenerative response. Two mouse models of stem cell injury will be studied, including the well-established 12 Gy gamma-irradiation model and a new model of CBC loss resulting from acute Notch inhibition that we define in this proposal. Our preliminary findings show that these two injuries both result in rapid CBC loss followed by a hyperproliferative regenerative response associated with a surge of Notch pathway signaling. Our preliminary studies also show that IGF-1 expression is induced by injury and that mTORC1 signaling is required for crypt repair. The proposed studies will: (1) identify specific Notch ligands required for the regenerative response, (2) test the role of Paneth cells in the regeneration response, and (3) test the role of IGF-1 and mTORC1 signaling for crypt repair and facultative stem cell mobilization. Furthermore, the studies will expand our knowledge of the intestinal stem cell niche, including definition of key niche cells in the epithelium and mesenchyme that respond to injury to regulate stem cell function. The studies take advantage of available genetic mouse models and pharmacologic tools to manipulate the signaling pathways under study to probe their function in adult mice. Understanding mechanisms regulating intestinal stem cell expansion and crypt regeneration is important to identify key strategies to propagate adult stem cells in culture for regenerative therapies as well as to develop treatment strategies for intestinal diseases associated with mucosal injury.